Genetic, pharmacological and imaging studies point to altered dopamine (DA) signaling as a risk determinant of Attention-deficit Hyperactivity Disorder (ADHD) and ADHD comorbid disorders. The presynaptic DA transporter (DAT, SLC6A3) has drawn particular interest due to its interactions with the most commonly prescribed ADHD medications, methylphenidate (MPH, Ritalin) and amphetamine (AMPH) formulations (e.g. Adderall). MPH is a DAT antagonist, whereas AMPH competes with DA for transport by DAT and induces DAT-mediated, non-vesicular DA release. The molecular/cellular basis for the effectiveness of these agents in ADHD treatment, and their relationship to a DA-based etiology of ADHD is unclear, owing in large part to a lack of understanding of the etiology of the disorder. This fact has also contributed to a lack of animal models with construct validity, necessitating the use of models with trait similarities (e.g., hyperactivity) but where origins likly have little or no relationship to disease mechanisms. The genetic risk for neuropsychiatric disorders, including ADHD, is believed to arise from contributions of both common and rare genetic variation. Whereas common genetic variation is often difficult to model, owing to a lack of penetrance and conservation across species, rare variation, particularly polymorphisms that impact protein coding sequence, offers the prospect of targeting conserved elements of proteins to achieve functional changes in molecules, cells and circuits. Recently, we identified a rare, coding variant (Val559) in the SLC6A4 gene of two male siblings with ADHD, a variant also identified in subjects with bipolar disorder and autism. Our studies of DAT Val559 expressed in transfected cells and neurons revealed that although DAT protein expression and DA uptake are normal, the variant displays a striking, voltage-dependent, DAT-mediated, anomalous DA efflux (ADE) phenotype. Remarkably, AMPH suppresses the ADE of DAT Val559, whereas it induces DA efflux from WT DAT (Ala559). DAT Val559 is also phosphorylated at N-terminal sites that are normally phosphorylated only after AMPH application, and the mutation of these sites in DAT Val559 to preclude phosphorylation eliminates ADE. These studies led us to the novel hypothesis that tonic DA leak, arising from perturbations of a DAT regulatory network that normally sustains concentrative DA uptake, leads to DA-linked traits prominently featured in multiple neurobehavioral disorders. To test this hypothesis, we have generated DAT Val559 knock-in mice and initiated a multi-disciplinary program to profile their biochemical, physiologica and behavioral features. Our Preliminary Studies provide evidence of ADE in vivo that leads to anomalous actions of local and systemically administered AMPH. In the current proposal, we seek to extend these studies to establish molecular, physiological and behavioral perturbations that arise in the model, studies that can redirect the field toward a novel framework for RDoc-responsive diagnoses and treatments underlying multiple psychiatric disorders.